Neuronal cell death is characteristic of most developing neural systems in vertebrates. The extent (30-80%) of neuronal death that occurs during development indicates that the regulation of this process is of fundamental importance to the determination of nervous system structure. Although a great deal of descriptive data has been reported concerning the magnitude and ubiquity of this neuronal cell loss, little is known of the mechanism that regulates this process during development. It is clear, however, that electrical activity plays an important role in determining neuronal survival during this regressive phase of development. It has been found, for example, blockage of electrical activity with .alpha.-bungarotoxin attenuates the naturally occurring cell death in spinal motoneurons (Pittman and Oppenheim, Nature (Lond.) 271, 364-366 (1987)) and in trochlear nucleic in vivo (Creazzo and Sohal, Exp. Neurol. 66, 135-145 (1979)).
Studies with cultured spinal cord-dorsal root ganglion (SC-DRG) neurons have shown that during development in vivo, neuronal cell death also occurs in a predictable and activity-dependent manner (Brenneman, et al., Peptides 6, 35-39 (1985)). Analysis of the effects of activity blockage on neuronal survival in culture has indicated an interaction between conditioning substances and electrical activity. When endogenous conditioning substances were removed before electrical blockade, neuronal cell death was accelerated (Brenneman, et al., Dev. Brain Res. 9, 13-27 (1983)). In contrast, when conditioning substances from SC-DRG cultures were supplied during blockage of electrical activity, neuronal cell death was prevented (Brenneman, et al., Dev. Brain Res. 15, 211-217 (1984)).
Further studies have indicated that part of the molecular basis of this activity-dependence is the action of vasoactive intestinal peptide (VIP), a neuropeptide which is released during electrical activity (Brenneman, D. E. and Eiden, L. E., Proc. Natl. Acad. Sci. U.S.A. 83, 1159-1162 (1986); and Brenneman, et al., Peptides 6, 35-39 (1985)). Moreover, studies have indicated that VIP increases the survival of activity-dependent spinal cord neurons by releasing protein growth factors from non-neuronal spinal cord cells (Brenneman, et al., J. Cell Biology, 104, 1603-1610 (1987)). More specifically, it has been determined that VIP interacts with its receptors on glial cells (Gozes, et al., Soc. Neurosci. Abs. 15, 216 (1989)) to induce the secretion of neuronal survival factor(s) (Brenneman, et al., J. Neurosci. Res. 25, 38&394 (1990); and Gozes, I. and Brenneman, D. E., Molecular Neurobiology, 3, 201-236 (1989)).
Among the growth factors released from non-neuronal spinal cord cells by VIP is Activity Dependent Neurotrophic Factor (ADNF). This glia-derived, VIP-released growth factor has been isolated from conditioned medium of rat cerebral cortical astroglia stimulated by VIP (Gozes, 1. & Brenneman, D. E., Molecular Neurobiology 3, 1-36 (1989); and Brenneman, D. B. & Eiden, L. E., Proc. Natl. Acad Sci. U.S.A. 83, 1159-1162 (1986)). Sequential chromatographic separations by ion exchange, gel permeation and hydrophobic interaction have been utilized to obtain about a 1650-fold purification of a single, 14,000 Dalton protein (apparent pI: 8.3.+-.0.25) that increases survival (EC50, 0.075 pg/ml) of electrically blocked spinal cord neurons and, accordingly, this glia-derived, VIP-released growth factor has been named: Activity Dependent Neurotrophic Factor. ADNF has been shown to protect neuronal cells against death. More particularly, ADNF has been shown to increase the growth and survival of developing spinal cord neurons, hippocampal neurons and cerebral cortical neurons. In addition, ADNF has been found to protect neuronal cell viability by preventing neuronal cell death produced by the external envelope protein of the HV virus.
Although ADNF effectively protects against neuronal cell death, it would be advantageous to have polypeptides which are shorter than the full length amino acid sequence of ADNF, but which exhibit the same neuroprotective/neurotrophic action of the intact ADNF growth factor. Quite surprisingly, the present invention provides such polypeptides.